US20040142482A1 - High-resolution ellipsometry method for quantitative or qualitative analysis of sample variations, biochip and measuring device - Google Patents

High-resolution ellipsometry method for quantitative or qualitative analysis of sample variations, biochip and measuring device Download PDF

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US20040142482A1
US20040142482A1 US10/478,574 US47857403A US2004142482A1 US 20040142482 A1 US20040142482 A1 US 20040142482A1 US 47857403 A US47857403 A US 47857403A US 2004142482 A1 US2004142482 A1 US 2004142482A1
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sample
biochip
metal layer
layer
measuring device
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Peter Westphal
Matthias Eberhardt
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Institut fuer Mikrotechnik Mainz GmbH
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/251—Colorimeters; Construction thereof
    • G01N21/253—Colorimeters; Construction thereof for batch operation, i.e. multisample apparatus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/21—Polarisation-affecting properties
    • G01N21/211—Ellipsometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G01N21/553—Attenuated total reflection and using surface plasmons

Definitions

  • the invention relates to a method for quantitative and/or qualitative determination of sample variations due to chemical, biological, biochemical or physical effects based on a change in the refraction index and/or the change in the layer thickness of the sample in accordance with claim 1 .
  • the invention further relates to the use of this method and to a corresponding measuring device in accordance with the preamble of claim 28 .
  • the invention also relates to a biochip in accordance with the preamble of claim 11 .
  • the resonance angle or the resonance wavelength is strongly influenced by the refraction index of the layer directly superjacent to the metal layer. If the resonance conditions change, e.g. because small amounts of water are replaced as a result of biological or chemical reactions while forming an additional layer, the minimum of the reflected intensity shifts. This shift can be used only to detect the qualitative growth of the layer but not its absolute thickness, because this would require knowing the refraction index of the growing layer. Thus, in addition to the substantial complexity of the apparatus, the measuring result is not very informative.
  • a corresponding measuring device is described, for example, in WO 90/05295.
  • the second method is ellipsometry.
  • the light is applied in such a way that it passes through a gaseous or liquid ambient medium and then strikes the biological or chemical layer to be detected (see H. Arwin, “Spectroscopic ellipsometry and biology: recent developments and challenges,” Thin Solid Films 313-314, 1998, pp. 764-774).
  • ⁇ essentially includes the change in intensity due to the reflection of the light.
  • ⁇ essentially includes the phase shift due to the reflection of the light; this parameter is very sensitive to layer thicknesses.
  • EP 0 067 921 discloses a method for determining bioactive substances using ellipsometric measurements.
  • a thin dielectric substrate is coated with an immobilization layer consisting of a first biologically active substance that interacts with a second bioactive substance.
  • Ellipsometric measurements are used to detect the optical changes in the biological layer.
  • the ellipsometric parameters are plotted as a function of time and these curves are compared with reference curves from measurements taken on biological material of known concentrations.
  • the sensitivity of the measurement obtained with radiation from the rear was 30 ⁇ poorer than with radiation from the front.
  • this prior art method has the drawback that special cuvettes must be used and titer plates cannot be used at all.
  • the unpublished German application DE 100 06 083.8 describes a method using an ellipsometric measurement in which the ellipsometric parameters ⁇ and ⁇ are established to determine quantitatively and/or qualitatively the layer thicknesses of the biological or chemical molecules being deposited due to interactions from a gaseous or liquid medium onto a metal film provided with an immobilization layer.
  • the angle of incidence and/or the frequency of the electromagnetic radiation used for the ellipsometric measurements are adjusted such that a surface plasmon resonance is produced in the metal layer.
  • the detection sensitivity ( ⁇ cos ⁇ /thickness of the layer to be determined) is adjusted via the thickness of the metal layer.
  • the electromagnetic radiation is directed onto the side of the metal layer opposite the immobilization layer.
  • At least one ellipsometric measurement is carried out during or after deposition and at least the corresponding cos ⁇ value is analyzed to determine the change in the thickness of the layer to be detected.
  • This method has the drawback that only individual samples can be tested. Testing of a large number of samples is time consuming because the individual samples must be successively brought into the beam path of the measuring device. This method cannot be used to test a plurality of samples simultaneously.
  • DNA and the RNA are of central importance.
  • the function of individual DNA sequences must first be determined. This requires, among other things, recognizing the differences between the DNA sequences of healthy and sick individuals.
  • the DNA strands can be placed, for example, using piezoelectric methods or can be synthesized directly on the chip surface using photolithographic methods.
  • hybridization reactions two individual, mutually complementary DNA or RNA strands form a double strand
  • it can be determined for example, where the differences occur between healthy and illness-inducing DNA fragments.
  • DNA and RNA fragments and proteins since DNA and RNA control protein production in cells, which is also referred to as transcription or translation.
  • So-called cDNA arrays are used to investigate the question as to which DNA is transcribed into mRNA.
  • the height per base pair is between approximately 0.2 nm and 0.4 nm, depending on the type of the helix.
  • the number of base pairs on DNA chips is usually 8 to 25, so that a strand height of approximately 2 to 8 nm is obtained.
  • the diameter of the helix is approximately 1.8 to 2.6 nm.
  • the average layer thickness can also be clearly below 1 nm, which requires a correspondingly sensitive detection method.
  • a so-called spot contains a certain number of DNA strands with an identical base sequence. Even for a comparatively small number of eight bases per strand, thousands of spots have to be applied to take into account all the possible base sequences. Detection of hybridization thus requires a sensitive measuring method that can be used to simultaneously analyze as many spots as possible.
  • Fluorescent labels have the drawback that they fade after a short time, which makes quantitative analyses more difficult.
  • highly sensitive low-noise CCD cameras are required for detection, which must be cooled to correspondingly low temperatures.
  • the base plate of such DNA chips is made, for example, of silicon. To accelerate the hybridization process of labeled samples, these chips may also be provided with microelectrodes.
  • a further object of the invention is to provide a corresponding measuring device for carrying out this method.
  • biochips adapted for use in this method and the measuring device.
  • the angle of incidence and/or the frequency of the electromagnetic radiation used for the ellipsometric measurements are set in such a way as to produce a damped surface plasmon resonance (SPR) in the metal film,
  • the detection sensitivity ( ⁇ cos ⁇ )/(sample variation unit) is adjusted via the thickness of the metal film
  • the electromagnetic radiation is directed two-dimensionally onto the side of the sample carrier opposite the sample and
  • At least two time-staggered, simultaneous, spatially resolved ellipsometric measurements are taken of the sample or samples and at least the corresponding ⁇ or cos ⁇ values are evaluated to determine the sample variation.
  • the invention is suitable for detecting all physical, chemical, biological or biochemical processes in which the refraction index near a surface changes sufficiently.
  • it is suitable for the above-described coupling reactions on a flat biochip, as will be described in greater detail below.
  • Sample variations should be understood to mean, in particular, the growth of layers of biological or chemical molecules deposited from a fluid onto an immobilization layer, particularly the initially described changes based on biochemical interactions, but also physical changes, such as, for example, the shrinkage or swelling of polymer films.
  • the ellipsometric parameter ⁇ is strongly influenced by surface plasmon excitation. If the wavelength and/or the angle of incidence of the electromagnetic radiation used is adjusted in relation to the employed metal such that a surface plasmon excitation occurs, the detection sensitivity is significantly increased and is on an order of magnitude greater than that afforded by conventional ellipsometry, i.e. without excitation of the surface plasmons.
  • the method according to the invention makes it possible to further increase the signal height and thus the detection sensitivity if the thickness of the metal film is optimized.
  • the thickness of the metal film By adjusting the thickness of the metal film, it is possible to increase the slope of the cos ⁇ curve and to increase the ratio of ⁇ cos ⁇ to sample variation. This may limit the dynamic range with respect to the maximum measurable sample variations since, in principle, the entire cos ⁇ change cannot be greater than 2. This is not a drawback, however, because if necessary the cos ⁇ change can also be reduced again via the selection of the film thickness or the light wavelength.
  • the spectral shift of the tan ⁇ and cos ⁇ curves is determined.
  • the relative and absolute change in the layer thickness or the refraction index can then be calculated.
  • the layer system to be examined is sufficiently known and is homogenous across the entire detection area, it is also possible to take the measurement at only a single wavelength.
  • the change in the tan ⁇ and cos ⁇ values is determined at a fixed wavelength. This assumes, however, that the additional growth in layer thickness or the change in the refraction index is not too large because the dynamic range of the cos ⁇ value is limited to ⁇ 1 to +1.
  • the cos ⁇ value proves to be the more sensitive value.
  • the advantage of the method according to the invention is that after setting the parameters for the surface plasmon excitation, it is not absolutely necessary to vary either the wavelength or the angle of incidence. This is a significant advantage with respective to the complexity of the apparatus as compared to measuring methods in which one of these parameters has to be varied.
  • the method according to the invention makes it possible to examine more samples per time unit than has heretofore been the case, because it is no longer necessary systematically to scan the angle of incidence and the wavelength.
  • the invention has the considerable advantage that it enables detection without labeling, making biochemical preparation easier and cheaper.
  • the biological interactions are not distorted by a fluorescent label.
  • Another significant advantage of the invention is that there are no fading effects as in fluorescence-based methods. This fading of the fluorescent dye is a well-known and significant problem in quantitative analyses of coupling reactions.
  • the method according to the invention does not require a highly sensitive camera because a significantly larger intensity component of the radiated light is available for detection.
  • the area of a biochip to be measured is relatively small—usually a few cm 2 —the entire chip can easily be covered by a single spatially resolved measurement.
  • the method is particularly suited for simultaneously detecting a plurality of biochemical coupling events.
  • a flat biochip provided with a metal coating is loaded with many different capture molecules. These capture molecules, which are immobilized on the metal layer, possibly using a so-called spacer (molecules to achieve favorable steric conditions), are capable of binding to very specific molecules from a solution. The resulting increase in mass on the surface can be measured by means of the associated change in the refraction index.
  • the capture molecules can be, for example, DNA fragments (oligonucleotides), antibodies, amino acid chains (peptides) as well as viruses or bacteria.
  • the capture molecules can be immobilized, for example, by means of streptavidin-biotin bonds, methods involving thiol chemistry or other wet chemical methods.
  • Another suitable method is to immobilize capture molecules on the titer-plate cuvette bottoms coated with a suitable metal film.
  • all the cuvettes are usually provided with the same type of capture molecules, but different solutions are subsequently placed into each of the cuvettes.
  • the entire titer plate can be measured using one image of the imaging sensor.
  • the simultaneous, spatially resolved ellipsometric measurements are conducted during as well as before and/or after the sample variations.
  • the measurements before the sample variation serve as reference measurements, which are compared with the measurement or measurements taken during or after the sample variation.
  • the change in cos ⁇ makes it possible to draw a conclusion regarding the magnitude of the sample variation.
  • the reference measurements can also be used for different samples.
  • Another preferred embodiment provides that continuous ellipsometric measurements are conducted at least during a time segment of the sample variations and that at least the change over time of the associated cos ⁇ value is analyzed. These measurements make it possible, for example, to track the growth of the layers to be detected.
  • the metal film used is made of a metal that has a refraction index (real part) of ⁇ 1 in the wavelength range of the electromagnetic radiation used.
  • the metal films used are ⁇ 50 nm.
  • thicknesses ranging from 10 to 45 nm, preferably between 10 and 40 nm have proven to be far more useful for the method according to the invention.
  • layer thicknesses ⁇ 50 nm the cos ⁇ curve is clearly flatter as a function of the radiated light frequency and the dynamic range of between ⁇ 1 and +1 is not exhausted.
  • metal layer thicknesses ⁇ 10 nm the sensitivities are insufficient.
  • the preferred thickness of the functional partial layer of between 20 nm and 40 nm ensures that the reflectivity collapse is reduced and spectrally broadened in surface plasmon resonance.
  • This physical behavior is referred to as damped surface plasmon resonance (damped SPR).
  • Damped surface plasmon resonance has the result that the ellipsometric values tan ⁇ and cos ⁇ do not change abruptly in case of wavelength variations.
  • This has the advantage that the spectral resolution of the entire measuring device can be relatively low, saving both costs and measuring time. As a rule, measuring at a few discrete wavelengths is sufficient to characterize the spectral shape of tan ⁇ and cos ⁇ with adequate precision.
  • the ellipsometric characterizability of the layers involved is an inherent advantage compared to non-ellipsometric SPR measuring devices. Preferably, all measurements are taken near the zero crossing of cos ⁇ on the wavelength scale because the detection sensitivity is greatest at this point.
  • electromagnetic radiation in the wavelength range of 100 nm to 10 ⁇ m, preferably 300 nm to 3 ⁇ m is used.
  • Monochromatic radiation, particularly light, is preferred.
  • the advantage of monochromatic radiation is that the radiation does not need to be spectrally filtered prior to detection.
  • Lasers may be used as radiation sources. It is also possible, however, to use lamps as a radiation source, e.g. xenon lamps with broad spectral distribution. In this case, spectral filtering is advantageously carried out prior to detection.
  • the method is preferably carried out on a biochip provided with a plurality of spots or on a plurality of microreaction vessels of a titer plate.
  • a preferred use of the method is the examination of biochemical interactions based on DNA or RNA hybridization, DNA or RNA protein interactions, DNA or RNA-antibody interactions or antibody-antigen interactions.
  • the method can be used for characterizing antibodies, developing immunoassays, optimizing ELISAs (ELISA: enzyme-linked immunoabsorbent assay), determining the concentration of small amounts of analyte, studying membranes or for investigating signal transduction chains.
  • ELISA enzyme-linked immunoabsorbent assay
  • the method is also suitable for examining physical or chemical sample variations in which the characteristics (complex refraction index, layer thickness, optical anisotropy, etc.) of thin films are spatially resolved.
  • the method can be used, for example, to investigate the shrinkage or swelling of polymer layers. It is also possible to determine the complex refraction index of liquids or polymerized solids. Further, the changes in concentration of ions, glucose or other ingredients in a liquid can also be determined.
  • the development over time of the diffusion process of soluble substances can be tracked with two-dimensional spatial resolution.
  • a biochip adapted for use with this method has a sample carrier with a base plate provided with at least one metal film.
  • the sample carrier is made of a material that has a transmission of at least 20% in the electromagnetic wavelength range of between 100 nm and 10 ⁇ m, at least in a wavelength segment having a width of at least 10 nm.
  • the metal film is preferably made of copper, silver, gold or aluminum alloy or an alloy containing at least 5 percent by weight of at least one of these metals.
  • the thickness of the metal layer, or the total thickness of several metal layers is between 10 and 45 nm, preferably between 20 and 40 nm.
  • Biochips are defined as DNA chips, RNA chips, electrophoresis chips and protein chips.
  • DNA or RNA chips also include the so-called DNA arrays, which are provided with a plurality of spots. DNA chips with only a single sample substance are also included.
  • the base plate of the sample carrier is preferably made of one of the materials BK7, SF10, SF11, ZrO 2 , fused silica, CrO 2 , Si 3 N 4 , quartz and/or a transparent plastic.
  • an adhesion promoting layer is disposed between the metal layer and the base plate.
  • This adhesion promoting layer significantly improves the adhesion of the functional metal layer on the transparent carrier.
  • This can be a sufficiently thin film of, for example, titanium or chromium.
  • the adhesion promoting layer is selected thin enough so that it does not interfere with the surface plasmon resonance excitation. Its thickness therefore ranges preferably from 1 nm to 20 nm.
  • a non-metallic cover layer may be applied to the metal layer, e.g. made of glass, metal oxide, semiconductor oxide and/or plastic. This layer is preferably no more than 500 nm thick.
  • the cover layer preferably has a transmission of ⁇ 10%.
  • a surface treatment with, for example, chemical solutions and/or plasmas can be used to adjust a hydrophilic or hydrophobic surface of the cover layer or the metal film.
  • a biochemical immobilization layer is applied to the metal film or the cover layer.
  • DNA spots are applied to the metal film or the cover layer.
  • the underside of the base plate advantageously carries a device for the two-dimensional coupling and decoupling of electromagnetic radiation.
  • a device for the two-dimensional coupling and decoupling of electromagnetic radiation can be, for example, a prism.
  • a trapezoidal prism can be made of one or more sections that can be bonded together, if necessary. The angle of incidence of the light changes as a function of the refraction index of the material used for the prisms.
  • the refraction index of the prism should largely correspond to that of the transparent base plate.
  • a medium should be introduced, the refraction index of which is likewise as similar as possible.
  • This can be an oil, another suitable liquid or a flexible solid. If a liquid or a viscous medium is used, it can be applied manually or by means of a pump device.
  • the metal layer or layers can be connected to a voltage source. In this case the metal layer also serves as an electrode.
  • the migration of charged particles in a liquid can be influenced, i.e. accelerated or impeded.
  • a counter-electrode may be provided at another site in the liquid containing the charged particles.
  • the electrodes can have electrical contact with the liquid or can be electrically isolated by non-conductive protective layers, e.g. made of SiO 2 .
  • the metal layer can also be applied partially so as to form a matrix-like structure.
  • the metallic matrix elements can each be connected to its own voltage source.
  • the matrix-like electronic structure can be configured in such a way that it is adapted to the matrix-like distribution of the DNA spots on a biochip.
  • the matrix-like, individually arranged electrodes can be supplied with individual leads and individual voltages.
  • the electrodes can also be electrically interconnected, such that only one voltage needs to be applied.
  • the measuring device comprises an ellipsometer, which has a radiation source, a polarizer, an analyzer and a detector as well as an evaluation unit connected to the detector.
  • the measuring device further comprises a sample carrier for the sample or samples to be measured, the base plate of which has at least one metal layer on the side facing the sample.
  • an optical coupling and decoupling device is arranged on the sample carrier. This coupling and decoupling device is configured in such a way that the electromagnetic radiation is directed onto the metal layer at an angle of incidence such that a damped surface plasmon resonance is excited.
  • a lens system each is arranged in the beam path, in front of and behind the coupling and decoupling device, which two-dimensionally illuminates the coupling and decoupling device and the detection surface of the detector.
  • the detector is an imaging sensor and thus enables the simultaneous spatially resolved measurement of the measurement signals.
  • the evaluation unit is configured for the spatially resolved simultaneous processing of the ( ⁇ cos ⁇ ) values.
  • the ellipsometer can be a zero ellipsometer, as it is described, for example, in Analytical Chemistry, Vol. 62, No. 17, Sep. 1, 1990, page 889. It can also be an ellipsometer with rotating polarizer or an ellipsometer with rotating analyzer or a phase-modulated ellipsometer.
  • the imaging sensor is preferably a CCD camera or a matrix-like arrangement of photodiodes or phototransistors.
  • the radiation source can be polychromatic, with a monochromator with variable wavelength or a filter wheel with optical band pass filters of different wavelengths being arranged between the radiation source and the imaging sensor.
  • the radiation source can also be a largely monochromatic light source or can consist of a plurality of largely monochromatic individual light sources with different wavelengths.
  • the lens system for two-dimensional radiation is preferably a Scheimpflug system.
  • a Scheimpflug system is advantageous for the sharp imaging of planes that are not parallel to the detection plane.
  • the coupling and decoupling device can be a prism made of BK7, SF10, SF11, ZrO 2 , fused silica, quartz or a transparent plastic.
  • the sample carrier can form the bottom of a reaction chamber.
  • the reaction chamber can have a temperature control and/or humidifying system.
  • biochips and sample carriers can also be transferred to titer plates.
  • FIG. 1 shows a measuring device according to the invention with a biochip
  • FIG. 2 shows a measuring device according to another embodiment
  • FIG. 3 shows a measuring device according to yet another embodiment with a titer plate
  • FIG. 4 is an enlarged detail of a biochip
  • FIG. 5 a is an enlarged detail of a microreaction vessel of a titer plate
  • FIG. 5 b is an enlarged detail of a microreaction vessel of a titer plate
  • FIG. 6 shows two diagrams to illustrate the adjustment of both the surface plasmon resonance and the thicknesses of the metal layer.
  • FIG. 7 shows the measurement of the changes in ⁇ cos ⁇ as a function of the wavelength of the radiated light
  • FIG. 8 shows ⁇ cos ⁇ as a function of the measuring time without metal film
  • FIG. 9 shows ⁇ cos ⁇ as a function of the measuring time using a silver film
  • FIG. 10 is a diagram showing ⁇ cos ⁇ over the measuring time during the hybridization process using a gold film
  • FIG. 11 is a three-dimensional bar diagram illustrating the spatially resolved measurements.
  • FIG. 1 is a schematic of a measuring device 1 .
  • the electromagnetic radiation 11 of a monochromatic light source e.g. a halogen lamp 2
  • a monochromatic light source e.g. a halogen lamp 2
  • the electromagnetic radiation 11 leaving the monochromator 4 is parallelized and if necessary expanded using an additional lens system 5 .
  • the monochromatic radiation is linearly polarized using a polarizer 6 and falls vertically onto the input surface 21 of a coupling and decoupling device 20 in the form of a prism.
  • the radiation passes through the input surface 21 with low reflection losses and negligible refraction and falls onto a further prism surface.
  • a thin oil film for adapting the refraction index is disposed between this prism surface and the sample carrier 30 located thereon.
  • the transparent carrier 30 is made of a homogenous glass or plastic material and has a refraction index that is as close as possible to that of the coupling and decoupling device 20 .
  • the radiation After the radiation has passed through the base plate 31 of the sample carrier 30 , it is reflected on the metal film 33 and because of the excitation of a damped surface plasmon resonance is weakened in its intensity and changed with respect to its phase or polarization.
  • the reflected radiation strikes a rotating analyzer 7 used to determine the reflection-associated intensity and phase changes for the s- and p-components (components polarized perpendicular and parallel to the plane of incidence) of the radiation.
  • the radiation then passes through a lens system 8 , preferably a Scheimpflug system, by means of which it is imaged on the imaging sensor 9 in the form of a CCD camera.
  • the imaging sensor 9 relays its signals to an evaluation and control device 10 , which further processes the signals and also coordinates the entire measurement process.
  • the ellipsometric measuring device is used to analyze a biochip 40 with matrix-like DNA spots 41 with different base sequences.
  • the DNA spots 41 are immobilized on the metal layer 33 and surrounded by an aqueous solution.
  • the aqueous solution can be replaced via an inflow 61 and an outflow 62 .
  • an agitator 65 with an associated drive is provided to accelerate hybridization processes or other biological interactions.
  • the aqueous liquid can be adjusted to a fixed temperature or can be cooled or heated during the measurement using a temperature control system 63 .
  • the ellipsometric values tan ⁇ and ⁇ cos ⁇ are determined spatially resolved for a plurality of radiation wavelengths in the range of the damped surface plasmon resonance.
  • the strength of the biological interactions at the different DNA spots 41 is determined from the ellipsometric measurement data.
  • FIG. 2 shows a measuring device according to another embodiment, which is distinguished from the arrangement depicted in FIG. 1 in that the DNA spots 41 on the biochip 40 are not surrounded by an aqueous but by a gaseous medium, e.g. air, nitrogen or argon. Because the refraction index of gaseous media is low compared to aqueous media, a small angle of incidence is provided for the electromagnetic radiation, so that a damped surface plasmon resonance can be excited in the same spectral range. Biochemical substances are generally more stable in a gaseous environment if the humidity is high. A humidifying system 66 is therefore provided in addition to the temperature control system.
  • a gaseous medium e.g. air, nitrogen or argon.
  • FIG. 3 shows yet another embodiment of a measuring device 1 , which is distinguished from the measuring device depicted in FIG. 1 in that a titer plate 50 with a matrix-like arrangement of indentations (cuvettes 55 ) is analyzed instead of a biochip 40 . Because of the large dimensions of the titer plate, the prism and all other components are likewise made correspondingly larger.
  • the cuvettes 55 are filled with a liquid.
  • the remaining temperature-controlled space is filled with a gaseous medium.
  • a humidifying system 66 is again provided in addition to the temperature-control system.
  • FIG. 4 is an enlarged detail of an area of a biochip 40 in the region of a spot 41 .
  • the layer structure of the biochip 40 consists of a base plate 31 , an adhesion promoting layer 32 , a metal layer 33 , a cover layer 34 , an immobilization layer 51 and one or more spots 41 applied thereto.
  • the base plate 31 can be, e.g., an ordinary microscope slide.
  • the base plate 31 is typically about 1 mm thick.
  • the refraction index of the base plate is adjusted to that of the prism of the coupling and decoupling device 20 .
  • the adhesion-promoting layer 32 e.g. made of titanium or chromium, is between 1.5 and 15 nm thick.
  • the 20 nm to 30 nm thick metal layer 33 is made of gold and is applied to the adhesion-promoting layer 32 .
  • the thickness of the gold layer 33 is a special feature that distinguishes this biochip from other gold-coated biochips.
  • Conventional gold-coated biochips usually have a gold layer of 50 nm or more.
  • a gold layer thickness of between 20 and 30 nm is optimal.
  • the metal layer or layers are preferably applied by vapor deposition or sputtering.
  • DNA spots 41 there is a matrix-like arrangement of DNA spots 41 with different base sequences. There can be up to 500,000 DNA spots 41 per cm 2 .
  • the DNA strands are immobilized on the chip, e.g. by injecting droplets (spotting), by photolithography or by using the phosphoramide method.
  • the DNA spots can be provided with a soluble biochemical protective layer that protects them against denaturation.
  • FIG. 5 a shows a titer plate 50 and an enlarged detail in the area of a microreaction vessel 55 .
  • the titer plate 50 is distinguished from a conventional, commercially available titer plate in that the inside of the microreaction vessels 55 of the base plate 31 is provided with a gold layer 33 that is applied to an adhesion promoting layer 32 .
  • the adhesion promoting layer is 1.5 nm to 15 nm thick, while the gold layer is 20 nm to 30 nm thick.
  • the transparent bottoms of the microreaction vessels can be made of plastic or glass.
  • the thickness of the base plate 31 typically ranges from 0.1 mm to 1 mm.
  • the bottoms are either a fixed component of a titer plate molded from plastic or parts of a glass or plastic plate that is bonded to a titer plate without bottom.
  • the refraction index of the bottoms is adjusted to that of the prism of the coupling and decoupling device.
  • the underside of the titer plate, which is placed onto the coupling and decoupling device, is unstructured and smooth.
  • the layer 51 is thus an immobilization layer.
  • the different microreaction vessels can contain the same or different capture molecules. These capture molecules are, for example, antibodies, single strand DNA, proteins, peptides or more complex structures, such as viruses or bacteria. For storage, the capture molecules can be provided with a soluble biochemical protective layer that protects them against denaturation. During measurement, the microreaction vessels contain a liquid.
  • FIG. 6 shows tan ⁇ and the lower part cos ⁇ , each as a function of the radiated wavelength.
  • the unpolarized light is directed onto the underside of the bottom wall of a cuvette at an angle of incidence of, e.g., 70°, as shown in FIG. 5 b by way of example.
  • a distinct minimum which is associated with a steep edge of the corresponding cos ⁇ curve, is established for tan ⁇ at a specific wavelength.
  • Metal layer thicknesses ⁇ 10 nm For metal layer thicknesses of ⁇ 10 nm, however, the resulting sensitivities are rather too low. Metal layer thicknesses ⁇ 50 nm are less suitable for the method according to the invention because of the small dynamic range. Only the curves for the thicknesses of 20 to 40 run show a steep slope and thus high detection sensitivity, with the entire dynamic range between ⁇ 1 and +1 being utilized.
  • FIG. 7 shows cos ⁇ as a function of the wavelength of the radiated light.
  • the solid curve on the left shows measurements without antibodies while the dotted curve indicates measurements with antibodies.
  • the measurements were taken on cuvettes with glass bottoms whose bottom wall is provided with a 12 nm thick titanium layer, a 27 nm thick silver layer and a 17 nm thick streptavidin layer as the immobilization layer.
  • the angle of incidence of the light is 70°. After an interaction period of 10 min, a 2.5 nm thick antibody layer grows, which is detected by the shift of the cos ⁇ curve.
  • the spectral measurements serve to determine the optimal wavelength with respect to the detection sensitivities of the dynamic range.
  • an optimal wavelength range of 640 to 700 nm resulted. If a single-wavelength measurement is taken at e.g. 680 nm, an increase in the thickness of the antibody layer approximately proportional to the change in the cos ⁇ value can be measured as a function of the incubation time of the antibody solution. After an increase in the antibody layer by 2.5 nm, the cos ⁇ value has changed by approximately 0.2. The resulting detection sensitivity (
  • FIG. 8 shows cos ⁇ as a function of the measuring time. This is a comparison measurement in which the metal layer on the bottom wall of the cuvette was absent. The arrows mark the instants when an aqueous buffer solution or an antibody-containing aqueous solution was used at a concentration of 66 pmol/ml and, respectively, 223 pmol/ml (see FIG. 8).
  • FIG. 10 shows a diagram illustrating the detection of DNA hybridizations.
  • the detected individual strands of the DNA molecules have a mass of approximately 6 k dalton and are thus clearly smaller and more difficult to detect than, for example, antibodies (typically 150 k dalton).
  • the determined thickness of the hybridized DNA layer of approximately 2 nm leads to a cos ⁇ change of 0.2.
  • Such a large ratio of the cos ⁇ change to the change in layer thickness is achieved with no other known ellipsometric device.
  • the cos ⁇ change of 0.2 was above the detection limit by approximately a factor of 100. With an optimized ellipsometer it is possible to achieve even lower detection limits and thus greater sensitivity.
  • the value 0.25 was deducted from the tan ⁇ scale for reasons of representation.
  • FIG. 11 is a three-dimensional representation of a simultaneous spatially resolved measurement.
  • a simultaneous, spatially resolved measurement was taken on a titer plate ( 1536 format). The number of the simultaneously measured cuvettes was 12.
  • the adhesion-promoting layer was made of 10 nm thick titanium.
  • the metal layer was 25 nm gold.
  • the bar diagram shows a differentiation measurement upon a change in the ion concentration:
  • the bar height corresponds to cos ⁇ 1 -cos ⁇ 2 .
  • the corresponding change in the refraction index in the solution was 0.004.
  • the wavelength used was 680 nm and the angle of incidence was 70°.
  • the individual bars in the diagrams are associated with individual cuvettes of the titer plate.
  • the measurement shows that the method according to the invention can be used for simultaneous spatially resolved measurements of small changes in the refraction index (in this case a liquid).

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